Characterization and discovery of novel miRNAs and moRNAs in chronic myeloproliferative neoplasms
Stefania Bortoluzzi1, Andrea Bisognin1, Marta Biasiolo1, Paola Guglielmelli2, Flavia Biamonte2, Ruggero Norfo3, Rossella Manfredini3, Alessandro M. Vannucchi2 on behalf of the AGIMM Investigators
1Department of Biology, University of Padova, Padova, Italy
2Department of Hematology, University of Florence, Florence, Italy
3Centre for Regenerative Medicine, Department of Biomedical Sciences, University of Modena and Reggio Emilia, Modena, Italy
Chronic myeloproliferative neoplasms (MPN) including polycythemia vera (PV), essential thrombocythemia (ET), primary myelofibrosis (PMF), and myelofibrosis post-PV (PPV_MF) are relatively common hematologic disorders showing clonal proliferation of hematopoietic stem cell and overproduction of mature blood cells, often associated with a JAK2 mutation. Reduced life expectancy is due to thrombosis, myeloproliferation or leukemia. In the frame of an ongoing molecular oncology project integrating experimental, systems biology and clinical approaches with the aim of developing innovative diagnostic/prognostic/therapeutic strategies, we exploited Illumina deep sequencing of short RNAs to characterize the miRNome in samples from MPN patients and in SET2 cells (JAK2V617F mutated cell line derived from a MPN patient). A novel computational framework for characterization of RNAs expressed from known miRNAs loci was developed and applied to characterize SET2 cells miRnome. 652 known mature miRNAs were detected, of which 21 were highly expressed, thus being responsible of most of miRNA-mediated gene repression. microRNA putative targets were enriched in specific signaling pathways, providing information about cell activities under massive post-transcriptional regulation. The majority of miRNAs were mixtures of sequence variants, called isomiRs, mainly due to alternative, non-canonical processing of hairpin precursors. We also identified 78 novel miRNAs (miRNA*) derived from known hairpin precursors. Both major and minor (*) form of miRNAs were expressed concurrently from half of expressed hairpins, highlighting the relevance of miRNA* and the complexity of strand selection bias regulation. Finally, we discovered that SET2 cells express a number of miRNA-offset RNAs (moRNAs), short RNAs derived from genomic regions flanking mature miRNAs. We provided novel data about the possible origin of moRNAs, while their functional role remains to be elucidated. Evidence indicates that moRNAs, initially considered by-products of miRNA processing machinery, may be a new class of functional RNAs, whose role(s) need to be clarified, to reveal if and how qualitative and quantitative alterations of moRNAs may impact on human disease. The identification of abnormally expressed miRNA and moRNas in CD34+ cells from patients with MPN, as well as the identification of disease-specific isomiRs or isomiRs mixtures, will allow to explore the usefulness of novel miRNome information to improve current criteria for therapeutic intervention, disease categorization and prognostication.
